1. Field of the Invention
The present invention relates to the field of the detection of microorganisms in the growth phase.
The present invention will find its application primarily in the areas of the industrial and clinical microbiology, for example in the pharmaceutical, biotechnological, agro-food industries or also in the hospitals or medical analysis laboratories.
The invention relates more particularly to a device permitting an incubation and quick detection of the forming of colonies, from microorganisms present in a sample, at the surface of a membrane or a solid or semi-solid culture medium.
2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98
Many techniques are presently implemented to permit the detection of contaminants, for example bacteria, in a sample to be tested.
The most conventional and oldest method consists of a deposition on the surface of an agar growth medium, whereby the latter may be more or less selective for one or several types of microorganisms.
The medium is then incubated at the appropriate temperature for the growth of the micro-organism(s) looked for, for a period that can be of up to several days.
Such a method has the drawback of requiring a relatively long incubation period in order to permit a detection with the naked eye of the colonies that were formed on the culture medium.
It is also known from the prior art to perform a polymerization chain reaction, also referred to as PCR, in order to determine the presence of specific microorganisms within a sample, by amplifying a DNA or RNA sequence.
These methods have the disadvantage of requiring several DNA strands, i.e. several contaminating microorganisms, generally at least several dozens of microorganisms. Such methods are therefore often less sensitive than the growth-based methods.
Also known is the possibility of using techniques consisting in marking the microorganisms so as to emphasize the contrast between the light emitted by the microorganisms and that emitted by the growth medium. The use of fluorescent viability markers such as CFDA (carboxyfluorescein diacetate) or non-fluorescent viability markers such as the TTC (tetrazolium chloride), or also of enzymes permitting to reveal the bioluminescence emitted for example by the ATP (adenosine triphosphate), permit an early detection of microorganisms, thanks to the use of optical systems sensitive to the characteristics of the emitted light, for example the wavelength or intensity.
Thus known is namely the United States patent application US 2003/0155528 that describes a method for detecting microorganisms, in which the latter are marked by suitable fluorescent reagents (such as fluorescein) permitting, on the one hand, to determine the amount microorganisms and, on the other, to judge whether they are viable or dead cells.
However, these techniques can prove cumbersome to be implemented and require the use of often expensive reagents and the presence of skilled labor forces. Moreover, they are not very suitable for detecting contaminants on a large number of samples, the marking operation being often difficult to be automated. In addition, these techniques have a risk of contamination of the sample. Indeed, the addition of reagents requires contacting said reagent with the microorganisms to be detected and is generally performed by opening the box containing the agar medium. Finally, contacting the reagent with the microorganisms has the risk of destructing the living cells forming a colony, especially when they are in early stages of growth (typically less than 100 cells).
From the prior art are also known methods based on a use of the light properties emitted naturally by microorganisms, for example by detecting the auto-fluorescence of said microorganisms. Thus, it is possible to facilitate the distinction of colonies using the contrast existing between the natural fluorescence emitted by said microorganisms and the non-fluorescent medium, on which they have been deposited.
A method using this principle is namely described in the international patent application WO 03/022999, in which certain optical properties of the colonies, such as auto-fluorescence, are used.
These techniques indeed permit to facilitate the detection of auto-fluorescent colonies or microorganisms, they then have a better contrast with the membrane or the culture medium. However, the level of naturally generated fluorescence is of a low magnitude, which does not permit to obtain fast detection times compared to a marking with a specific fluorophore, for example. In addition, the spurious emission of natural fluorescence by the culture medium or other particles present in the environment, such as dust, membrane fibers, or plastic particles from the medium, can cause a false positive result.
Finally, techniques using optical systems with high magnification can be used to visualize colonies in early stages of development: this is the case with microscopes, for example. However, these devices are limited to a detection on small surfaces, generally smaller than 1 mm2. Thus, the implementation of this type of techniques for the detection of one or more detection media, such as membranes or agar media, proves both long, of about several minutes per cm2, and expensive, due to the necessity of using scanning systems.
Also known from the prior art is patent WO 2013/110734, which provides a device for an early detection, and which can be automated, of the appearance of colonies on the surface of a growth medium, namely a membrane or an agar culture medium.
More particularly, in this device can be found a detection surface, on which rests a growth medium, for example a membrane or an agar medium, and a detection system, such as a linear scanner. This system includes at least one CCD sensor associated with an optical system.
This device is particularly interesting, because it namely permits to omit the use of expensive optical equipment or reagents. In addition, such a device permits to avoid any contamination by other microorganisms or particles, because the samples do not need to be moved during incubation, since the detection system is mobile.
However, it has been found that such a detection device, though very performant per se, could be further improved in its performance. It was also found that the developments made to such a detection device could also improve the performances of the incubation and detection devices that do not use a linear scanner, for example.